RU2457844C2 - Antiviral medication and method of prevention and treatment of viral infections - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to field of veterinary. Claimed is application of antiviral medication sodium salt of 2-methyltio-6-nitro-1,2,4-triazolo[5,1-c]-1,2,4-triazin-7-on dihidrate for prevention and treatment of viral diseases and states induced by Hantaan virus or virus of eastern equine encephalomyelitis. Claimed is method of treating viral diseases and states induced by Hantaan virus or virus of Eastern equine encephalomyelitis by introduction of said medication.

EFFECT: group of inventions is efficient for treatment, prevention and urgent prophylaxis of Hantaan virus or virus of Eastern equine encephalomyelitis.

21 cl, 3 tbl

Description

The claimed invention relates to medicine and pharmacy, in particular to the treatment, prevention and prevention of viral diseases, mainly caused mainly by the Huntaan virus or eastern encephalomyelitis in horses (hereinafter - VESEL).

Analysis of the changing environmental conditions of the virus circulation in nature, the evolution of antiviral drugs increasingly convincingly indicate that one of the fundamental approaches to the problem of prevention and treatment of viral infections should be an analysis of viral pathology as a complex dynamic process that requires constant adjustment of the strategy and tactics of combating viral infections infections.

For certain socio-political reasons, the epidemic danger of the introduction of pathogens of dangerous and especially dangerous viral infections and their spread on the territory of our country increases (see, for example, Pokrovsky V.I., Onishchenko G.G., Cherkassky B.Ya. Actual directions improvement of the prevention of infectious diseases // Epidemiol. and inf. illnesses. - 2000. - No. 1. - C.4-8).

One of the serious public health problems is natural focal viral infections, among which the leading hemorrhagic fevers are leading (see, for example, Chu J.-K., Jennings GB, Schmaljohn CS A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul virus but not Puumala virus // J. Virol. - 1995. - Vol.69, No. 10. - P.6417-6423; Dekonenko A.E., Tkachenko EA, Lipskaya G.Yu. and others Genetic differentiation of hantaviruses by polymerase reaction and sequencing // Vopro virol. - 1996. - No. 1. - P.24).

Hemorrhagic fever with renal syndrome (HFRS) is an acute viral natural focal infection of a person, characterized by polymorphism of clinical manifestations involving almost all organs and systems in the pathological process, severe course and the development of serious complications. The disease proceeds with severe infectious-toxic and hemorrhagic manifestations, kidney damage and the development of acute renal failure. Recently, HFRS has become increasingly important in human pathology and is one of the leading places among other natural focal infections.

HFRS takes a leading place among zoonotic viral infections and one of the first places among all natural focal human diseases. On the territory of the Russian Federation is one of the largest and most active global HFRS foci. In the Russian Federation, 61 of 89 administrative territories are endemic to HFRS. Moreover, 97% of cases were detected in the European part of the Russian Federation and 3% in the Asian part, mainly in the Far East (see, for example, Tkachenko E.A., Drozdov S.G., Fedorov Yu.M. Hantaviruses and hantavirus infections // Collection of abstracts of the scientific conference of the IPVE “Actual problems of medical virology”, Moscow, November 23–25, 1999 - M., 1999. - T.2. - P.88).

Annually, approximately 200 thousand cases of HFRS disease are recorded in the world, more than 500 strains of hantaviruses have been isolated - 120 of them in the territory of the former Soviet Union (see, for example, Shcherbakova S.A., Popov N.V., Doblo A.D. and etc. Some results and prospects of studying the circulation of HFRS virus in the Saratov region // Collected scientific papers “The Problem of Particularly Dangerous Infections.” - Saratov, 2000. - Issue No. 80. - P.48-52).

All of the above determines the relevance of the study of new antiviral drugs for the prevention and treatment of topical viral infections. The lack of effective nonspecific antiviral drugs for natural focal viral infections of the Russian Federation stimulates the intensification of work on the synthesis of new compounds of different chemical structures.

Chemotherapy has much less contraindications than vaccines. For storage, mobile refrigerators (refrigerators) are not required and there is no need to conduct bioassays before direct use. In this case, the use of nonspecific drugs for people with impaired immunity is shown. Thus, chemotherapeutic agents are an alternative means of treating and preventing dangerous and especially dangerous viral infections, especially infections for which specific protective agents have not been developed.

Long-term studies have shown that the best antiviral agents either selectively inhibit individual stages of viral reproduction or act indirectly through the host cell. A very promising group of antiviral drugs are abnormal nucleosides and their derivatives (see, for example, Ershov F.I. Medicines against viruses // Materia Medica (bull. For doctors and pharmacists). - 1996. - No. 2 (10). - C .5-13).

To date, tactics for studying the effectiveness of chemotherapy drugs for a number of viral infections are described in the literature (see, for example, Votyakov VI, Borenko EI An approach to assessing the activity of chemical compounds against viruses propagating in tissue cultures // Methodological issues of scientific development antiviral substances. - Minsk, 1977. - S.10-14; Chizhov NP. Experimental methods and criteria for evaluating the inhibitory activity of antiviral substances. - Minsk, 1977. - C.36-41).

The preclinical study of the drug consists of several stages: the study of the effectiveness of drugs in cell culture and in a laboratory model that reproduces the leading syndrome of the disease and has the similarity of the clinic, the pathogenesis of infection in humans.

The difficulty of studying hantavirus infections is noted, since this is associated with the search for an experimental form of infection in animals. An analysis of the literature indicates that this problem has so far not had a positive solution.

One of the promising antiviral drug compounds for the treatment of the Huntaan virus or VESEL is the sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1-s] -1,2,4-triazin-7-one dihydrate (hereinafter - the Means).

A biologically active compound is known from the prior art that protects against infections caused by Rift Valley fever viruses, as well as active against the ZEL virus, parainfluenza, respiratory syncytial virus, infectious bird laryngotracheitis, a total of 10 RNA and DNA containing viruses (see patent for Invention No. 2294936 “Sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo [5.1-s] -1,2,4-triazin-7-one dihydrate having antiviral activity,” submission date 29.06 .2005, published March 10, 2007).

To date, the strategy for selecting effective non-specific medical remedies for a number of viral infections is described in the Guidelines for the preclinical evaluation of drugs (see the Guidelines for the experimental (preclinical) study of new pharmacological substances. - M., Ministry of Health of the Russian Federation, 2005).

The technical result, the achievement of which the claimed invention is directed, is to expand the assortment of medicines with a wide spectrum of action and intended for the treatment, prevention and emergency prevention of the Huntaan virus or VESEL.

The specified technical result is achieved by the fact that for the treatment, prevention and emergency prevention of infectious diseases and conditions of viral etiology caused by the Huntaan or VSEL viruses, an antiviral drug is used - sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo [ 5.1-c] -1,2,4-triazin-7-one dihydrate.

In this case, the disease or condition is hemorrhagic fever with renal syndrome (caused by the Huntaan virus) or VESEL. The drug is intended for systemic administration using a dosage amount, for example, in an amount of 50 mg / kg, for the prevention of disease and emergency prevention.

The drug, made in the form of a solution, tablets or capsules, is intended for administration to humans, domestic or farm animals orally or in the form of injections.

Technical solutions that coincide with the totality of the essential features of the claimed invention have not been identified, which allows us to conclude that the claimed invention meets such a patentability condition as “novelty”.

The claimed essential features that predetermine the receipt of the specified technical result, do not explicitly follow from the prior art, which allows us to conclude that the claimed invention meets such a patentability condition as "inventive step".

The patentability condition "industrial applicability" is confirmed by examples of specific performance.

Studies to evaluate the effectiveness of the drug in relation to the Hantaan or VSEL viruses in comparison with the control in cell culture and laboratory animals were carried out in the following areas:

- assessment of the maximum tolerated dose (MTD);

- assessment of antiviral efficacy of HFRS agents in the monolayer of the culture of transplantable pig embryonic kidney cells (SPEV) to suppress the level of virus reproduction;

- the choice of a laboratory model for studying the effectiveness of the Means in relation to experimental forms of HFRS;

- Evaluation of the effectiveness of drugs on laboratory animals infected with HFRS and horse encephalomyelitis pathogens using emergency prevention and treatment regimens.

Statistical processing of the results was carried out by generally accepted methods (see Lakin GF Biometrics. - M.: Higher School. - 1990).

Hantaan virus (the causative agent of HFRS) and horse eastern encephalomyelitis virus were used in the work.

Two-week-old white sucker mice were used to simulate the lethal form of HFRS; white mice weighing 8-10 g were used to simulate the experimental forms of VESL.

Monitoring of infected animals was carried out for 21 days, while the death of animals was monitored twice during the day.

Toxicity Means were evaluated on uninfected two- and four-week-old mice.

The main criterion for evaluating the effectiveness was the indicator of animal protection against death (percentage) during in vivo tests and the suppression of virus reproduction during in vitro tests. The following criteria served as additional criteria: average life time (days) and the level of suppression of virus reproduction in brain tissues (Δ, log).

Example 1

Evaluation of the effectiveness of the Agent against the Hantaan virus was carried out in a culture of SPEV cells to suppress the reproduction of the studied pathogens in the presence of chemotherapeutic agents. The level of virus accumulation in cell culture was evaluated by titration using the method of formation of negative colonies.

A two-day monolayer of SPEV cell culture was infected with a virus at a dose of 0.02 PFU / cell at a temperature of (37 ± 0.5) ° С. For the Huntaan virus, the adsorption time was 90 minutes. After adsorption, the monolayer of cells was washed with 3 volumes of maintenance medium, fresh medium with various concentrations of the Agent (from 1 to 100 μg / ml) was introduced into the tubes. For each dose of the Agent, 4 tubes with a monolayer of cells were used. Incubation was carried out at (37 ± 0.5) ° C for the Huntaan virus for 7 days. At the end of the incubation period, cryodestruction of the cells was carried out and the level of virus accumulation was determined by the formation of negative colonies in the GMK-AN-1 (D) cell culture for the Huntaan virus. The research results are presented in table 1.

Table 1 Evaluation of antiviral efficacy In vitro agents against the Huntaan virus (HFRS agent) The concentration of the drug, mcg / ml Means virus titer, lgBOE / ml titer reduction, Δ, lg one hundred 3.0 2.0 fifty 3,5 1,5 twenty 3.8 1,2 10 3.8 1,2 5 3.9 1,1 one 4.8 0.2 Control (without drug) 5,0 -

From the data presented in table 1, it can be seen that only the agent in a sufficiently high concentration significantly suppresses the reproduction of the Hantaan virus (a 100-fold decrease in the reproduction of the virus was observed).

Example 2. The study of the inhibitory effect of funds on the causative agent of HFRS was carried out on white sucker mice 12 days old, infected subcutaneously with the Hantaan virus, at a dose of 6 lgBOE.

The effectiveness of the drug was evaluated by the clinical manifestations and death of animals, the titer of the virus was determined by the method of I.P. Ashmarin et al. (see Ashmarin I.P., Vasiliev N.N., Ambrolov V.A. Fast methods of statistical processing and planning of experiments. - L., 1975. - 103 p.).

Taking into account the magnitude of the acute toxic dose, the working dose of the Agent was chosen - 50 mg / kg of animal weight.

Evaluation of the effectiveness of funds was carried out according to the following schemes:

1. Scheme of emergency prevention - the animals were injected with a virus-containing suspension in a volume of 0.1 ml subcutaneously. The agent was administered 2 hours after infection orally and then once a day for 5 days at a working dose.

2. Treatment schedule - the animals were injected with a virus-containing suspension in a volume of 0.1 ml subcutaneously, 24 hours after infection, the Means were administered orally and then once a day for 4 days in a test dose.

The results of evaluating the antiviral effectiveness of the Means when administered orally to outbred white sucker mice infected subcutaneously with the Hantaan virus are presented in Table 2.

table 2 Antiviral efficacy Remedies for an experimental form of hantavirus infection A drug Scheme of drug administration The number of dead mice Average life time, day Protection against death, percent Decrease in the level of virus accumulation in the brain, Δ, lg Means +2, +24, 48, 72, 96, 120 18/22 11.7 16.7 5.0 1.8 Control (without drug) - 32/32 8.5 - 6.8 - Herd control - 0/20 21.0 - -

It should be noted that in the experimental group observed a significant lengthening of the life span.

From the data presented in table 2, it is seen that the use of funds for emergency prevention more effectively inhibits the reproduction of the virus in the brain tissue. For Means, the suppression ratio was almost 100.

Thus, lengthening the life span of animals in the experimental group and decreasing the level of virus accumulation in the brain indicate a positive effect of the studied Medication on the dynamics of the infectious process when using drugs for emergency prevention of experimental hantavirus infection.

Example 3

To study the antiviral effect of the Agent in relation to the experimental form of VES in white mice (infection dose of 10 LD ₅₀ ), the administration scheme 1 and 2 were used.

Scheme 1. The drug was administered to mice orally at a dose of 50 mg / kg 2 hours after infection and then daily for 5 days once a day.

Scheme 2. The drug was administered to mice orally at a dose of 50 mg / kg 24 hours after infection and then daily for 4 days once a day.

Table 3 presents the results of a study of the effectiveness of the Agent in relation to the experimental form of VES in white mice with parenteral infection.

Protective efficacy of the Agent in relation to the experimental form of VESEL in white mice when administered to animals according to the emergency prevention scheme was 50.0%; the lengthening of the average life time in the group of animals was 7.2 days.

Table 3 Evaluation of antiviral activity Means in relation to the experimental form of VSEL A drug Scheme The number of animals in the experiment, pcs The number of dead animals, pcs Protection against death, percent Life expectancy, day Suppression of virus reproduction in the brain, Δ, lg Means No. 1 thirty 8 73.3 16.9 7.1 1.3 Number 2 thirty 9 70.0 15.7 7.2 1,2 Dose control 36 36 - 3.9 8.3 - Herd control thirty 0 - - - Placebo control thirty 0 - - -

The tool showed high protective efficacy against the experimental form of VES in white mice when administered to animals according to the emergency prevention scheme, protection against death was 73.3%; the lengthening of the average life time in the group of animals was 13.0 days.

In the group of treated animals, the average lifetime was significantly increased. The tool showed high protective effectiveness (73.3%) in relation to the experimental form of VESEL.

Thus, the drug - sodium salt of 2-methylthio-6-nitro-1,2-4-triazolo [5.1-s] -1,2,4-triazin-7-one dihydrate during the study is moderately effective against Hantaan virus at a dose of 50 mg / kg. When using the Means according to emergency prevention and treatment regimens, it has high protective efficacy against VES pathogens when administered orally at a dose of 50 mg / kg.

The claimed invention helps to expand the range of medicines with a wide spectrum of action and intended for the treatment, prevention and emergency prevention of viral infections, in particular, caused by the Huntaan virus or eastern encephalomyelitis of horses (hereinafter - VESEL).

Claims (21)

1. The use of antiviral agents sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5.1-s] -1,2,4-triazin-7-one dihydrate (Means) for the prevention and treatment of viral Diseases and conditions caused by the Hantaan virus or equine encephalomyelitis virus. 2. The use according to claim 1, characterized in that the disease or condition is hemorrhagic fever with renal syndrome or equine encephalomyelitis. 3. The use according to claim 1, characterized in that the tool is intended for systemic administration. 4. The use according to claim 1, characterized in that a metered amount of the drug is used. 5. The use according to claim 1, characterized in that the tool is intended for administration to humans, domestic or farm animals. 6. The use according to claim 1, characterized in that the agent is administered orally. 7. The use according to claim 1, characterized in that the tool is administered in the form of injections. 8. The use according to claim 6, characterized in that for the prevention and treatment of diseases, the amount of the agent in each dosage amount is 50 mg / kg. 9. The use according to claim 1, characterized in that the tool is made in the form of tablets. 10. The use according to claim 1, characterized in that the tool is made in the form of capsules. 11. The use according to claim 1, characterized in that the tool is made in the form of an injection form. 12. A method for the prevention and treatment of conditions caused by the Hantaan or eastern encephalomyelitis viruses of horses in humans or animals, characterized in that it involves the administration of a drug - sodium salt of 2-methylthio-6-nitro-1,2,4-triazolo [5,1 -c] -1,2,4-triazin-7-one, dihydrate. 13. The method according to p. 12, characterized in that it is intended for the prevention and treatment of diseases. 14. The method according to p. 12, characterized in that the disease is a hemorrhagic fever with renal syndrome or eastern encephalomyelitis in horses. 15. The method according to p. 12, characterized in that the disease is caused by the Huntaan virus or the virus of eastern encephalomyelitis in horses. 16. The method according to p. 12, characterized in that the introduction of funds carry out a metered amount. 17. The method according to p. 12, characterized in that the tool is administered orally. 18. The method according to clause 16, characterized in that for the treatment and emergency prevention of the disease, the amount of the drug in each dosage amount is 50 mg / kg 19. The method according to p. 12, characterized in that the tool is made in the form of tablets or in the form of capsules. 20. The method according to p. 12, characterized in that the tool is administered in the form of injections. 21. The method according to p. 12, characterized in that the tool is made in the form of an injection form.